1. Field of the Invention
The present invention relates to a fluorescence polarization method at multiple wavelengths for analyzing two or more different assay-objects in a sample in one reaction system. The present invention is particularly useful in the fields of art of environmental assay, food control, and medical diagnosis.
2. Description of the Related Art
The fluorescence polarization method is known in the art as a method for assaying a substance in a sample. The method is based on the principle that when a fluorescent-labeled compound is excited by linearly polarized light, the fluorescence emitted from the compound has a degree of polarization which is in proportion to the molecular weight thereof.
As a fluorescence polarization method which has been developed, there is a fluorescence polarization immunossay based on an antigen-antibody reaction.
For example, U.S. Pat. No. 4,902,630 discloses an assay method in which a body fluid (particularly, a blood) containing CRP, an assay-object, is added to a mixed solution which contains: a xe2x80x9ctracerxe2x80x9d obtained by binding fluorescein, a fluorochrome, to C-reactive protein (CRP); and an antibody which is capable of specifically binding to CRP. CRP in the sample is assayed based on competition between the tracer and CRP for binding the antibody in the mixed solution.
Japanese Laid-Open Publication No. 3-188374 discloses a method in which a fluorescent-labeled particle comprising an insoluble carrier particle carrying a fluorochrome (or a phosphorescence dye) and an antigen (or an antibody) is reacted with a sample solution containing an antibody (or an antigen). The antibody (or an antigen) in the sample is assayed based on the aggregation of the particles due to an antigen-antibody reaction.
However, the conventional methods are designed to assay only one substance in a sample, and cannot assay two or more different substances in a sample in one reaction system. Accordingly, there has been a demand in the art for development of a method which is suitable for assaying two or more different substances in a sample in one reaction system.
According to one aspect of this invention, there is provided a fluorescence polarization method at multiple wavelengths for analyzing two or more different assay-objects in a sample. The method includes the steps of: (a) providing two or more different fluorescent-labeled substances, each being a substance which is capable of specifically binding to respective one of the assay-objects and is covalently bound to a fluorochrome, wherein the fluorochromes of the fluorescent-labeled substances are different from one another; (b) allowing the fluorescent-labeled substances to bind to the two or more different assay-objects, respectively; and (c) measuring a change in the degree of fluorescence polarization which has taken place in each of the fluorescent-labeled substances by its binding to one of the assay-objects.
In one embodiment of the invention, each of the two or more different assay-objects is independently a biological substance, a microorganism, a virus, a pharmaceutical, an environmental pollutant or an abused drug.
In one embodiment of the invention, the biological substance is a peptide, a protein, a lipid, a saccharide or a nucleic acid.
In one embodiment of the invention, the protein is an antibody, a hormone, an inflammation marker, a coagulation factor, an apolipoprotein, a high density lipoprotein (HDL), a low density lipoprotein (LDL), a glycosylated albumin, a glycosylated hemoglobin, a hemoglobin, or an enzyme.
In one embodiment of the invention, the hormone is chorionic gonadotropin, thyroid-stimulating hormone, progesterone, follicular forming hormone, parathyroid-stimulating hormone, adrenocorticotropic hormone, or insulin.
In one embodiment of the invention, the inflammation marker is C-reactive protein (CRP), xcex11-antitrypsin (xcex11-AT), xcex1l-antichymotrypsin (xcex1l-X), xcex1l-acid glycoprotein (xcex11-AG), haptoglobin (Hp), ceruloplasmin (Cp), the 9th component of complement (C9), the 4th component of complement (C4), the 3rd component of complement (C3), complement factor B (B), fibrinogen (Fbg), serum amyloid A (SAA), C1 inhibitor (ClI), a sialoglycoprotein, an acid-soluble protein (ASP) or an immunosuppressive acidic protein (IAP).
In one embodiment of the invention, the microorganism is staphylococcus, Sarcina, Spirillum, Steptococcus, coccobacillus, bacillus, Spirochaeta, tetracoccus, comma bacillus, or Actinomyces.
In one embodiment of the invention, the specifically-binding substance is a protein which is a antibody, an antigen, a receptor, or an inhibitor.
In one embodiment of the invention, the antibody is a set of polyclonal antibodies, a monoclonal antibody, a chimeric antibody, a Fab antibody or a (Fab)2 antibody.
In one embodiment of the invention, the fluorochrome has a functional group which is capable of binding to a primary, secondary or tertiary amino group, a carboxyl group, a thiol group, a phenyl group, a phenol group or a hydroxyl group.
In one embodiment of the invention, a lifetime of fluorescence of the fluorochrome is in the range of about 0.1 nanoseconds to about 500 nanoseconds.
In one embodiment of the invention, the fluorochrome has a skeletal structure of fluorescein, dansyl, pyrene, rhodamine, dialkylaminonaphthalene, dialkylamnionaphthalenesulfonyl, cyanin, or indolenine.
In one embodiment of the invention, each of the fluorochromes is selected so that the fluorochrome is different from the other fluorochromes in terms of at least one of excitation wavelength, fluorescence wavelength, and lifetime of fluorescence.
According to another aspect of this invention, there is provided a kit for use in a fluorescence polarization method at multiple wavelengths for analyzing two or more different assay-objects in a sample. The kit includes two or more different fluorescent-labeled substances, each being a substance which is capable of specifically binding to respective one of the assay-objects and is covalently bound to a fluorochrome, wherein the fluorochromes of the fluorescent-labeled substances are different from one another.
According to still another aspect of this invention, there is provided a system for use in a fluorescence polarization method at multiple wavelengths for analyzing two or more different assay-objects in a sample. The system includes: (a) two or more different fluorescent-labeled substances, each being a substance which is capable of specifically binding to respective one of the assay-objects and is covalently bound to a fluorochrome, wherein the fluorochromes of the fluorescent-labeled substances are different from one another; and (b) means for measuring the degree of fluorescence polarization.
In the assay system according to the method of the present invention, a change in molecular weight which has taken place in each fluorescent-labeled substance by its binding to an assay-object is measured as a change over time in the molecular orientation. Thus, it is possible to analyze, in a single process, two or more different assay-objects having different molecular weights, by appropriately selecting the types of fluorochromes used as labels in view of the change in molecular weight before and after the binding of fluorochromes.
Thus, the invention described herein makes possible the advantages of (1) providing a fluorescence polarization method at multiple wavelengths for analyzing two or more different assay-objects contained in a sample in one reaction system by measuring a change in the degree of fluorescence polarization for each of the assay-objects; (2) providing a kit for analyzing two or more different assay-objects contained in a sample by using a fluorescence polarization method at multiple wavelengths; and (3) providing a system for analyzing two or more different assay-objects contained in a sample by using a fluorescence polarization method at multiple wavelengths.